Figure 2
Ptp4E expression and protein localization during embryogenesis. (a-f) Expression of Ptp4E during development was visualized by in situ hybridization of whole-mount embryos. In all panels, anterior is left and dorsal is up, except for (c), which shows a ventral view. (a) At stage 8, in gastrulating embryos, Ptp4E expression is enriched in the mesoderm (labelled m). (b,c) In germ band extended stage 11 embryos, Ptp4E is expressed at highest levels in the posterior midgut primordium (PMG) and also shows a scalloped pattern that corresponds to the visceral mesoderm (vm). (d) As a positive control, we also stained embryos where UAS-Ptp4E-GFP was driven using the engrailed-GAL4 driver. The characteristic striped engrailed pattern is observed, indicating that the probe recognizes Ptp4E transcripts. (e) At stage 15, the strongest Ptp4E signal is in the midgut (mg); the ventral nerve cord (CNS) is also visible (arrow). (f) At stage 17, the gut is segmented and starts to coil. Ptp4E is expressed at highest levels at the anterior and posterior ends of the midgut and in the hindgut (hg). (g-o) Anti-Ptp4E antibodies recognize ectopically expressed Ptp4E-GFP. (g,h) The UAS-Ptp4E-GFP construct was ectopically expressed using the engrailed-GAL4 driver in stage 11 embryos. Anti-Ptp4E signal (g; red) colocalizes with theUAS-Ptp4E-GFP (visualized with anti-GFP; h, green) expression pattern, indicating that the antibody specifically recognizes the Ptp4E protein. (i) In wild-type embryos, the antibody shows a low and ubiquitous signal. Ptp4E-GFP protein was expressed in all postmitotic neurons using elav-GAL4 (j-o). Stage 16 embryo was visualized with anti-Ptp4E (j,m), anti-GFP (k,n), and merged (l,o). Ptp4E-GFP protein is transported out to the axons (m-o) as the CNS ladder brightly stains with anti-Ptp4E and anti-GFP. Ptp4E also accumulates in cell bodies, since the CNS region outside of the axon tracts stains brightly (j,k,m,n, arrows). PNS cell bodies (chordotonal organs) are also visible (j,k, arrowheads). (j-l) Images are projections of confocal stacks. (m-o) Images are single confocal sections. Scale bars are 35 microns (a-i) and 20 microns (j-o).